Syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a second outbreak significantly delaying ... more Syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a second outbreak significantly delaying the hope for the virus’ complete eradication. In the absence of effective vaccines, we need effective treatments with low adverse effects that can treat hospitalized patients with COVID-19 disease. In this study, we determined the existence of SARS-CoV-2-specific T cells within CD45RA– memory T cells in the blood of convalescent donors. Memory T cells can respond quickly to infection and provide long-term immune protection to reduce the severity of COVID-19 symptoms. Also, CD45RA– memory T cells confer protection from other pathogens encountered by the donors throughout their life. It is of vital importance to resolve other secondary infections that usually develop in patients hospitalized with COVID-19. We found SARS-CoV-2-specific memory T cells in all of the CD45RA– subsets (CD3+, CD4+, and CD8+) and in the central memory and effector memory subpopulations. The procedure for obtaining...
Dietary composition plays an important role in the pathophysiology of type 2 diabetes. Monounsatu... more Dietary composition plays an important role in the pathophysiology of type 2 diabetes. Monounsaturated fatty acid consumption has been positively associated with improved insulin sensitivity and β-cell function. We examined whether an extra virgin olive oil (eVoo) high fat diet (HFD) can improve glucose homeostasis. C57BL/6J mice were fed a standard diet or a lard-based HFD to induce type 2 diabetes. Then, HFD mice were fed with three different based HFD (lard, EVOO and EVOO rich in phenolic compounds) for 24 weeks. HFD-EVOO diets significantly improved glycemia, insulinemia, glucose tolerance, insulin sensitivity and insulin degradation. Moreover, eVoo diets reduced β-cell apoptosis, increased β-cell number and normalized islet glucose metabolism and glucose induced insulin secretion. No additional effects were observed by higher levels of phenolic compounds. thus, eVoo intake regulated glucose homeostasis by improving insulin sensitivity and pancreatic β-cell function, in a type 2 diabetes HFD animal model. The occurrence of metabolic diseases is rapidly increasing worldwide, and major risk factors for these diseases include being overweight or obese. Considering the increased incidence of these illnesses, the rise in type 2 diabetes (T2D) and nonalcoholic fatty liver disease (NAFLD) is particularly worrying. Lifestyle changes are responsible for this increase, especially a reduction in physical activity and chronic overnutrition 1,2. Dietary composition plays an important role in the pathophysiology of T2D and NAFLD. Dietary fats are of particular interest, with regard to not only the amount of fats but also the type of fats 3,4. In recent years, several studies have researched the influence of various dietary fats on T2D; however, their impact is still not fully understood. In addition, the underlying mechanisms of these fats are not clear. The proposed hypothesis is that dietary fats primarily modify the fatty acid composition of the cell membrane, thereby altering cellular functions related to insulin sensitivity 5. Some studies have shown that feeding rats with different dietary fats affect endocrine pancreas structure and function 6,7. Regarding pancreatic β-cells, the lipotoxicity hypothesis states that an excess of free fatty acids (FFAs) affects β-cell survival and insulin secretory functions 8. Nevertheless, little is known about the effects of the different FFAs on β-cells. Data from controlled intervention studies indicate that replacing saturated fatty acids (SFAs) and trans FFAs with monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) reduces the risk of T2D 3. In addition, a cross-sectional study of the general population in Spain revealed a favorable relationship of a MUFA-rich diet with β-cell secretion of insulin 9. Most studies have assessed the effects of dietary fatty acids on the prevention of metabolic diseases; however, exploring the ability of certain FFAs to reverse problems in glucose homeostasis may be more clinically relevant.
s / Transplant Cell Ther 28 3S (2021) S1–S509 S217 potential as well as the phenotypic and functi... more s / Transplant Cell Ther 28 3S (2021) S1–S509 S217 potential as well as the phenotypic and functional characteristics of post-REP TIL were then evaluated. Results: While DAC treatment led to a decrease in TIL expansion and an increase in the CD4+ to CD8+ ratio, this coincided with an increase in the frequency of central memory-like cells (CD45RA-CCR7+) as well as the expression of IL-7R and the transcription factors TCF1, Eomes, and KLF2, suggesting a shift towards a more memory-like phenotype. Additionally, DAC treatment increased the expression of CD25, CD28, and ICOS while reducing the expression of inhibitory receptors like PD-1 and TIGIT. Following stimulation, DAC-treated TIL showed increased degranulation and a higher frequency of IFN+TNF+ cells, which translated into increased cytotoxicity. Conclusions: DAC treatment during TIL expansion can shift the balance away from effector differentiation and towards a more memory-like phenotype, while conferring increased expression of co-stimulatory receptors, reduced expression of inhibitory markers and improved functionality. Inhibiting DNA methylation programs during TIL expansion could represent a useful approach for modifying the epigenetic landscape of TIL, imprinted prior to ex vivo expansion and introduced during the expansion process itself, to improve their therapeutic potential.
Abstract Many dairy products found in plastics, cosmetics and food containers disrupt human healt... more Abstract Many dairy products found in plastics, cosmetics and food containers disrupt human health due to their actions as endocrine-disrupting chemicals (EDCs). Studies in different cellular and animal models have reported that low doses of the EDC bisphenol A (BPA), modify pancreatic β-cell function, induce insulin resistance and other metabolic alterations. Here, we aimed to investigate whether administration of low doses of BPA could regulate ion channels expression and function in mice pancreatic islets. For this purpose, we used microarrays to analyze the global gene expression profile, focusing on the modulation of ion channels-related genes. For in vivo experiments, mice were subcutaneously injected with BPA (100 μg/kg/day during 4 days). Islets were isolated 12 h after the last injection and prepared for RNA extraction and hybridization on Affymetrix GeneChip Mouse Genome 430 2.0 Array. Array scanning and data analysis were performed using Affymetrix Expression Console and the Affymetrix Transcriptome Analysis Console (TAC) Softwares. Expression level analysis was performed using the normalization method based on the processing algorithm called robust multi-array average (RMA). Whole islets or dispersed islets from C57BL/6 or estrogen receptor beta knockout (βERKO) mice were treated in vitro with BPA or vehicle during 48 h. Whole-cell patch-clamp recordings were used to measure sodium and potassium currents. Microarray analysis of islets isolated from mice treated with BPA showed that some ion channels, such as sodium channel voltage-gated type IX, alpha (Scn9a), potassium large conductance calcium-activated channel alpha member 1 (Kcnma1), and Kv channel-interacting protein 1 (Kcnip1), were modulated. These results were confirmed by quantitative RT-PCR. Electrophysiological measurements showed a decrease in both sodium currents and total potassium currents. Additionally, the currents through the voltage-gated potassium channel subunits Kv2.1 and 2.2 as well as the KCa1.1 (encoded by Kcnma1 gene) channels were also reduced. Interestingly, beta-cells from βERKO mice did not present such reductions, suggesting that these effects occur mainly via ERβ. Our results show that, acting as a xenoestrogen, BPA modulates sodium and potassium currents as well as gene expression via ERβ. Funding: Ministerio de Economia y Competitividad, Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER), EU Grants SAF2014-58335-P, BFU2017-86579-R, BFU2016-77125-R and Generalitat Valenciana PROMETEO II/2015/016. LM holds a Juan de la Cierva fellowship from the Ministry of Economy, Industry and Competitiveness (IJCI-2015-24482). CIBERDEM is an initiative of the Instituto de Salud Carlos III. J-AG was supported by the Robert A. Welch Foundation (E-0004).
Recent developments in the effort to understand the metabolism and function of the intracellular ... more Recent developments in the effort to understand the metabolism and function of the intracellular dinucleoside polyphosphates were described by nine speakers from some of the world's leading laboratories in this field in a workshop at the Purines 2000 International Symposium on Nucleosides and Nucleotides held in Madrid in July, 2000. Topics were wide-ranging and included phenotypic analyses of yeast mutants defective in enzymes of dinucleoside polyphosphate degradation, virally encoded catabolic enzymes, the structure and function of the Fhit tumor suppressor and Fhit-nitrilase fusion proteins and the relationship of Fhit to human diadenosine triphosphate hydrolase, site-directed mutagenesis of diadenosine tetraphosphate hydrolase, novel nucleotide analogs for studying hydrolase function, the synthesis of dinucleoside polyphosphates by ligases, and the possible roles of diadenosine tri-and tetraphosphates in insulin function and of diadenosine tetraphosphate in the heat-shock response of Escherichia coli. The results presented and the ensuing discussions showed that, while considerable progress is being made in the field, it still has the capacity to tease and frustrate and produce the unexpected result. Drug Dev. Res.
... ACTH-Like Peptides and Their Modulatory Effect on lnsulin Secretion 121 Juan J. Gagliardino, ... more ... ACTH-Like Peptides and Their Modulatory Effect on lnsulin Secretion 121 Juan J. Gagliardino, Maria 1. Borelli, Fernando Estivariz, 1llani ... Mediators in Cytokine-1nduced Pancreatic P-Cell Dysfunction and Destruction 313 Michael L. McDaniel, John A. Corbett, Guim Kwon, and ...
The effects of a lipid component of oxidized low-density lipoproteins (ox-LDL), L-α-palmitoyllyso... more The effects of a lipid component of oxidized low-density lipoproteins (ox-LDL), L-α-palmitoyllysophosphatidylcholine (LPC), on membrane currents of isolated canine renal artery smooth muscle cells (RASMC) were examined using the whole-cell configuration of the patch-clamp technique. In RASMC exposed to nominally Ca 2+-free solutions and dialyzed with 0.1 mM EGTA and 140 mM K + , superfusion with LPC (10 µM) elicited spontaneous transient outward currents (STOCs) and/or spontaneous transient inward currents (STICs), followed by the activation of a large voltage-independent current with a reversal potential (E r) close to 0 mV. Buffering intracellular Ca 2+ with 10 mM BAPTA prevented the appearance of STOCs and STICs, but not the activation of the voltage-independent current. E r of the LPC-induced voltage-independent current exhibited sensitivity to changes in [K + ] o and [Na + ] o in a manner consistent with a non-selective cation current (I NSC) and was blocked by gadolinium (Gd 3+ ; 10 µM). Shifts in E r of the LPC-induced I NSC in response to changes in [Ca 2+ ] o were used to estimate a relative Ca 2+ to Na + permeability ratio (P Ca /P Na) of 1.67. These results suggest that LPC causes abnormal sarcoplasmic reticulum Ca 2+ regulation, leading to the appearance of STOCs and STICs, and the activation of I NSC in vascular smooth muscle cells. These effects may explain the ability of ox-LDLs to elevate [Ca 2+ ] i in vascular smooth muscle and inhibit endothelium-dependent relaxation.
It has been recently demonstrated (Ferrer et al., 1984) that the transition element zinc blocks b... more It has been recently demonstrated (Ferrer et al., 1984) that the transition element zinc blocks both glucoseinduced electrical activity and insulin release from mouse pancreatic islets. Zn2+ is present in the insulin secretory granules and is released with insulin into the islet extracellular space. The levels of free Zn2+ in the extracellular space surrounding 0-cells may be sufficient to affect islet function. The mean plasma concentration in normal mice was found to be 74 k 7pM (20 animals). Therefore, the observation reported may be of physiological importance. Previous data from Ferrer e f al. (1984) permits us to postulate that ZnZ+ inhibits voltage-dependent CaZ+ channels. We here report further evidence that ZnZ+ acts in this way.
Allogeneic islet cell transplantation is currently the only curative treatment of diabetes mellit... more Allogeneic islet cell transplantation is currently the only curative treatment of diabetes mellitus. However due to organ shortage islet transplantation is limited and immunsupressive protocols are hazardous and complicated side effects. The incidence of diabetes is constantly growing and a treatment with autologous insulin producing cells would hold great promise for the therapy of diabetes. We herein describe the generation of insulin producing »Neo-islet cells« from human peripheral blood monocytes, which are able to secret insulin in a glucose dependent fashion and normalize blood glucose levels after transplantation into diabetic mice for a period of ten days. In comparison with pancreatic islet cells »Neo-islet cells« exhibit a gene expression pattern which is comparable to an endocrine precursor cell as known from embryonic pancreas developmental studies.
Background: Adoptive cell immunotherapies for opportunistic virus in immunocompromised patients u... more Background: Adoptive cell immunotherapies for opportunistic virus in immunocompromised patients using haploidentical memory T cells have shown to be safe and effective. Since severe cases of COVID-19 present a dysregulated immune system with T cell lymphopenia and a hyper-inflammatory state we have proposed that a similar strategy could be proven to be efficient for COVID-19 patients. This is a study protocol of an open-label, multicenter, double-arm, randomized, dose-finding phase I/II clinical trial to evaluate the feasibility, safety, tolerability, and efficacy of the administration of a single dose of allogenic SARS-CoV-2 specific memory CD45RA - T cells and Natural Killer (NK) cells in COVID-19 patients with lymphopenia and pneumonia. The aim of the study is to find efficient treatments for patients with moderate/severe COVID-19. Identification of Specific memory T cells and NK cells: i)Memory T Cells: we first determined the existence of SARS-CoV-2 specific T cells within the CD45RA - T memory cells of the blood of convalescent donors. Memory T cells can respond quickly to the infection and provide long-term immune protection to reduce the severity of the COVID-19 symptoms without inducing classically T cell alloreactivity. Also, CD45RA - memory T cells confer protection for other pathogens the donors encountered in their life. ii)NK cells: we determined the phenotype of NK cells after COVID-19 and the main characteristic of SARS-CoV-2 specific NK population in the blood of convalescent donors, as it has been shown for cytomegalovirus infections. Also, NK cells confer protection for other pathogens the donors encountered in their life. Pilot Phase I- Safety, feasibility, and dose escalation: Between September and November 2020 a phase 1, dose-escalation, single-center clinical trial was conducted to evaluate the safety and feasibility of the infusion of CD45RA - memory T cells containing SARS-CoV-2 specific T cells as adoptive cell therapy against moderate/severe cases of COVID-19. Nine participants with pneumonia and/or lymphopenia and with at least one human leukocyte antigen (HLA) match with the donor were infused. The first three subjects received the lowest dose (1x10 5 cells/kg), the next three received the intermediate dose (5x10 5 cells/kg) and the last three received the highest dose (1x10 6 cells/kg) of CD45RA - memory T cells. Clinicaltrials.gov registration: NCT04578210. Findings: All participants' clinical status measured by National Early Warning Score (NEWS) and 7-category point ordinal scales showed improvement six days after infusion. No serious adverse events were reported. Inflammatory parameters were stabilized post-infusion and the participants showed lymphocyte recovery two weeks after the procedure. Donor microchimerism was observed at least for three weeks after infusion in all patients. Interpretation: This study provides preliminary evidence supporting the idea that treatment of COVID-19 patients with moderate/severe symptoms using convalescent SARS-CoV-2 specific CD45RA - memory T cells is feasible and safe. We did not find dose-liming toxicity. The Recommended Phase 2 dose was 1x10 6 CD45RA - T cells. Phase II- Efficacy: Between January 2021 and July 2021 patients have been enrolled based on the matched with the HLA genotype of the convalescent donors and following the protocol inclusion/exclusion criteria. The primary outcome is the incidence of patient recovery at day 14, defined as normalization of fever and oxygen saturation or lymphopenia recovery. Secondary outcomes are the time to normal level of lymphocytes, the proportion of patients showing clinical improvement at day 7, time to first negative SARS-CoV-2 PCR, the incidence of treatment-related adverse events, duration of hospitalization, time to discharge, time to improvement by one category a 7-point ordinal scale or NEWS score, the proportion of patients requiring intensive care unit, and all-cause mortality. In addition, lymphocyte recovery by multiparametric flow cytometry and donor chimerism by real-time PCR in the experimental arm was monitored weekly during the first month. This study provides preliminary evidence supporting the idea that treatment of COVID-19 patients with moderate/severe symptoms using convalescent CD45RA - memory T cells is safe and feasible. The phase II clinical trial is ongoing to demonstrate efficacy. Figure 1 Figure 1. Disclosures Soria: Celgene: Other: Fees; Gilead: Other: Fees; AbbVie: Other: Fees.
Aims/hypothesis Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical that has been ass... more Aims/hypothesis Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical that has been associated with type 2 diabetes development. Low doses of BPA modify pancreatic beta cell function and induce insulin resistance; some of these effects are mediated via activation of oestrogen receptors α (ERα) and β (ERβ). Here we investigated whether low doses of BPA regulate the expression and function of ion channel subunits involved in beta cell function. Methods Microarray gene profiling of isolated islets from vehicle-and BPA-treated (100 μg/kg per day for 4 days) mice was performed using Affymetrix GeneChip Mouse Genome 430.2 Array. Expression level analysis was performed using the normalisation method based on the processing algorithm 'robust multi-array average'. Whole islets or dispersed islets from C57BL/ 6J or oestrogen receptor β (ERβ) knockout (Erβ −/−) mice were treated with vehicle or BPA (1 nmol/l) for 48 h. Whole-cell patchclamp recordings were used to measure Na + and K + currents. mRNA expression was evaluated by quantitative real-time PCR. Results Microarray analysis showed that BPA modulated the expression of 1440 probe sets (1192 upregulated and 248 downregulated genes). Of these, more than 50 genes, including Scn9a, Kcnb2, Kcnma1 and Kcnip1, encoded important Na + and K + channel subunits. These findings were confirmed by quantitative RT-PCR in islets from C57BL/6J BPA-treated mice or whole islets treated ex vivo. Electrophysiological measurements showed a decrease in both Na + and K + currents in BPA-treated islets. The pharmacological profile indicated that BPA reduced currents mediated by voltage-activated K + channels (K v 2.1/2.2 channels) and large-conductance Ca 2+-activated K + channels (K Ca 1.1 channels), which agrees with BPA's effects on gene expression. Beta cells from ERβ −/− mice did not present BPA-induced changes, suggesting that ERβ mediates BPA's effects in pancreatic islets. Finally, BPA increased burst duration, reduced the amplitude of the action potential and enlarged the action potential halfwidth, leading to alteration in beta cell electrical activity.
Frontiers in Cell and Developmental Biology, Feb 15, 2023
Type 1 diabetes mellitus (T1DM) is an autoimmune disorder specifically targeting pancreatic islet... more Type 1 diabetes mellitus (T1DM) is an autoimmune disorder specifically targeting pancreatic islet beta cells. Despite many efforts focused on identifying new therapies able to counteract this autoimmune attack and/or stimulate beta cells regeneration, TD1M remains without effective clinical treatments providing no clear advantages over the conventional treatment with insulin. We previously postulated that both the inflammatory and immune responses and beta cell survival/regeneration must be simultaneously targeted to blunt the progression of disease. Umbilical cord-derived mesenchymal stromal cells (UC-MSC) exhibit anti-inflammatory, trophic, immunomodulatory and regenerative properties and have shown some beneficial yet controversial effects in clinical trials for T1DM. In order to clarify conflicting results, we herein dissected the cellular and molecular events derived from UC-MSC intraperitoneal administration (i.p.
Syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a second outbreak significantly delaying ... more Syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a second outbreak significantly delaying the hope for the virus’ complete eradication. In the absence of effective vaccines, we need effective treatments with low adverse effects that can treat hospitalized patients with COVID-19 disease. In this study, we determined the existence of SARS-CoV-2-specific T cells within CD45RA– memory T cells in the blood of convalescent donors. Memory T cells can respond quickly to infection and provide long-term immune protection to reduce the severity of COVID-19 symptoms. Also, CD45RA– memory T cells confer protection from other pathogens encountered by the donors throughout their life. It is of vital importance to resolve other secondary infections that usually develop in patients hospitalized with COVID-19. We found SARS-CoV-2-specific memory T cells in all of the CD45RA– subsets (CD3+, CD4+, and CD8+) and in the central memory and effector memory subpopulations. The procedure for obtaining...
Dietary composition plays an important role in the pathophysiology of type 2 diabetes. Monounsatu... more Dietary composition plays an important role in the pathophysiology of type 2 diabetes. Monounsaturated fatty acid consumption has been positively associated with improved insulin sensitivity and β-cell function. We examined whether an extra virgin olive oil (eVoo) high fat diet (HFD) can improve glucose homeostasis. C57BL/6J mice were fed a standard diet or a lard-based HFD to induce type 2 diabetes. Then, HFD mice were fed with three different based HFD (lard, EVOO and EVOO rich in phenolic compounds) for 24 weeks. HFD-EVOO diets significantly improved glycemia, insulinemia, glucose tolerance, insulin sensitivity and insulin degradation. Moreover, eVoo diets reduced β-cell apoptosis, increased β-cell number and normalized islet glucose metabolism and glucose induced insulin secretion. No additional effects were observed by higher levels of phenolic compounds. thus, eVoo intake regulated glucose homeostasis by improving insulin sensitivity and pancreatic β-cell function, in a type 2 diabetes HFD animal model. The occurrence of metabolic diseases is rapidly increasing worldwide, and major risk factors for these diseases include being overweight or obese. Considering the increased incidence of these illnesses, the rise in type 2 diabetes (T2D) and nonalcoholic fatty liver disease (NAFLD) is particularly worrying. Lifestyle changes are responsible for this increase, especially a reduction in physical activity and chronic overnutrition 1,2. Dietary composition plays an important role in the pathophysiology of T2D and NAFLD. Dietary fats are of particular interest, with regard to not only the amount of fats but also the type of fats 3,4. In recent years, several studies have researched the influence of various dietary fats on T2D; however, their impact is still not fully understood. In addition, the underlying mechanisms of these fats are not clear. The proposed hypothesis is that dietary fats primarily modify the fatty acid composition of the cell membrane, thereby altering cellular functions related to insulin sensitivity 5. Some studies have shown that feeding rats with different dietary fats affect endocrine pancreas structure and function 6,7. Regarding pancreatic β-cells, the lipotoxicity hypothesis states that an excess of free fatty acids (FFAs) affects β-cell survival and insulin secretory functions 8. Nevertheless, little is known about the effects of the different FFAs on β-cells. Data from controlled intervention studies indicate that replacing saturated fatty acids (SFAs) and trans FFAs with monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) reduces the risk of T2D 3. In addition, a cross-sectional study of the general population in Spain revealed a favorable relationship of a MUFA-rich diet with β-cell secretion of insulin 9. Most studies have assessed the effects of dietary fatty acids on the prevention of metabolic diseases; however, exploring the ability of certain FFAs to reverse problems in glucose homeostasis may be more clinically relevant.
s / Transplant Cell Ther 28 3S (2021) S1–S509 S217 potential as well as the phenotypic and functi... more s / Transplant Cell Ther 28 3S (2021) S1–S509 S217 potential as well as the phenotypic and functional characteristics of post-REP TIL were then evaluated. Results: While DAC treatment led to a decrease in TIL expansion and an increase in the CD4+ to CD8+ ratio, this coincided with an increase in the frequency of central memory-like cells (CD45RA-CCR7+) as well as the expression of IL-7R and the transcription factors TCF1, Eomes, and KLF2, suggesting a shift towards a more memory-like phenotype. Additionally, DAC treatment increased the expression of CD25, CD28, and ICOS while reducing the expression of inhibitory receptors like PD-1 and TIGIT. Following stimulation, DAC-treated TIL showed increased degranulation and a higher frequency of IFN+TNF+ cells, which translated into increased cytotoxicity. Conclusions: DAC treatment during TIL expansion can shift the balance away from effector differentiation and towards a more memory-like phenotype, while conferring increased expression of co-stimulatory receptors, reduced expression of inhibitory markers and improved functionality. Inhibiting DNA methylation programs during TIL expansion could represent a useful approach for modifying the epigenetic landscape of TIL, imprinted prior to ex vivo expansion and introduced during the expansion process itself, to improve their therapeutic potential.
Abstract Many dairy products found in plastics, cosmetics and food containers disrupt human healt... more Abstract Many dairy products found in plastics, cosmetics and food containers disrupt human health due to their actions as endocrine-disrupting chemicals (EDCs). Studies in different cellular and animal models have reported that low doses of the EDC bisphenol A (BPA), modify pancreatic β-cell function, induce insulin resistance and other metabolic alterations. Here, we aimed to investigate whether administration of low doses of BPA could regulate ion channels expression and function in mice pancreatic islets. For this purpose, we used microarrays to analyze the global gene expression profile, focusing on the modulation of ion channels-related genes. For in vivo experiments, mice were subcutaneously injected with BPA (100 μg/kg/day during 4 days). Islets were isolated 12 h after the last injection and prepared for RNA extraction and hybridization on Affymetrix GeneChip Mouse Genome 430 2.0 Array. Array scanning and data analysis were performed using Affymetrix Expression Console and the Affymetrix Transcriptome Analysis Console (TAC) Softwares. Expression level analysis was performed using the normalization method based on the processing algorithm called robust multi-array average (RMA). Whole islets or dispersed islets from C57BL/6 or estrogen receptor beta knockout (βERKO) mice were treated in vitro with BPA or vehicle during 48 h. Whole-cell patch-clamp recordings were used to measure sodium and potassium currents. Microarray analysis of islets isolated from mice treated with BPA showed that some ion channels, such as sodium channel voltage-gated type IX, alpha (Scn9a), potassium large conductance calcium-activated channel alpha member 1 (Kcnma1), and Kv channel-interacting protein 1 (Kcnip1), were modulated. These results were confirmed by quantitative RT-PCR. Electrophysiological measurements showed a decrease in both sodium currents and total potassium currents. Additionally, the currents through the voltage-gated potassium channel subunits Kv2.1 and 2.2 as well as the KCa1.1 (encoded by Kcnma1 gene) channels were also reduced. Interestingly, beta-cells from βERKO mice did not present such reductions, suggesting that these effects occur mainly via ERβ. Our results show that, acting as a xenoestrogen, BPA modulates sodium and potassium currents as well as gene expression via ERβ. Funding: Ministerio de Economia y Competitividad, Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER), EU Grants SAF2014-58335-P, BFU2017-86579-R, BFU2016-77125-R and Generalitat Valenciana PROMETEO II/2015/016. LM holds a Juan de la Cierva fellowship from the Ministry of Economy, Industry and Competitiveness (IJCI-2015-24482). CIBERDEM is an initiative of the Instituto de Salud Carlos III. J-AG was supported by the Robert A. Welch Foundation (E-0004).
Recent developments in the effort to understand the metabolism and function of the intracellular ... more Recent developments in the effort to understand the metabolism and function of the intracellular dinucleoside polyphosphates were described by nine speakers from some of the world's leading laboratories in this field in a workshop at the Purines 2000 International Symposium on Nucleosides and Nucleotides held in Madrid in July, 2000. Topics were wide-ranging and included phenotypic analyses of yeast mutants defective in enzymes of dinucleoside polyphosphate degradation, virally encoded catabolic enzymes, the structure and function of the Fhit tumor suppressor and Fhit-nitrilase fusion proteins and the relationship of Fhit to human diadenosine triphosphate hydrolase, site-directed mutagenesis of diadenosine tetraphosphate hydrolase, novel nucleotide analogs for studying hydrolase function, the synthesis of dinucleoside polyphosphates by ligases, and the possible roles of diadenosine tri-and tetraphosphates in insulin function and of diadenosine tetraphosphate in the heat-shock response of Escherichia coli. The results presented and the ensuing discussions showed that, while considerable progress is being made in the field, it still has the capacity to tease and frustrate and produce the unexpected result. Drug Dev. Res.
... ACTH-Like Peptides and Their Modulatory Effect on lnsulin Secretion 121 Juan J. Gagliardino, ... more ... ACTH-Like Peptides and Their Modulatory Effect on lnsulin Secretion 121 Juan J. Gagliardino, Maria 1. Borelli, Fernando Estivariz, 1llani ... Mediators in Cytokine-1nduced Pancreatic P-Cell Dysfunction and Destruction 313 Michael L. McDaniel, John A. Corbett, Guim Kwon, and ...
The effects of a lipid component of oxidized low-density lipoproteins (ox-LDL), L-α-palmitoyllyso... more The effects of a lipid component of oxidized low-density lipoproteins (ox-LDL), L-α-palmitoyllysophosphatidylcholine (LPC), on membrane currents of isolated canine renal artery smooth muscle cells (RASMC) were examined using the whole-cell configuration of the patch-clamp technique. In RASMC exposed to nominally Ca 2+-free solutions and dialyzed with 0.1 mM EGTA and 140 mM K + , superfusion with LPC (10 µM) elicited spontaneous transient outward currents (STOCs) and/or spontaneous transient inward currents (STICs), followed by the activation of a large voltage-independent current with a reversal potential (E r) close to 0 mV. Buffering intracellular Ca 2+ with 10 mM BAPTA prevented the appearance of STOCs and STICs, but not the activation of the voltage-independent current. E r of the LPC-induced voltage-independent current exhibited sensitivity to changes in [K + ] o and [Na + ] o in a manner consistent with a non-selective cation current (I NSC) and was blocked by gadolinium (Gd 3+ ; 10 µM). Shifts in E r of the LPC-induced I NSC in response to changes in [Ca 2+ ] o were used to estimate a relative Ca 2+ to Na + permeability ratio (P Ca /P Na) of 1.67. These results suggest that LPC causes abnormal sarcoplasmic reticulum Ca 2+ regulation, leading to the appearance of STOCs and STICs, and the activation of I NSC in vascular smooth muscle cells. These effects may explain the ability of ox-LDLs to elevate [Ca 2+ ] i in vascular smooth muscle and inhibit endothelium-dependent relaxation.
It has been recently demonstrated (Ferrer et al., 1984) that the transition element zinc blocks b... more It has been recently demonstrated (Ferrer et al., 1984) that the transition element zinc blocks both glucoseinduced electrical activity and insulin release from mouse pancreatic islets. Zn2+ is present in the insulin secretory granules and is released with insulin into the islet extracellular space. The levels of free Zn2+ in the extracellular space surrounding 0-cells may be sufficient to affect islet function. The mean plasma concentration in normal mice was found to be 74 k 7pM (20 animals). Therefore, the observation reported may be of physiological importance. Previous data from Ferrer e f al. (1984) permits us to postulate that ZnZ+ inhibits voltage-dependent CaZ+ channels. We here report further evidence that ZnZ+ acts in this way.
Allogeneic islet cell transplantation is currently the only curative treatment of diabetes mellit... more Allogeneic islet cell transplantation is currently the only curative treatment of diabetes mellitus. However due to organ shortage islet transplantation is limited and immunsupressive protocols are hazardous and complicated side effects. The incidence of diabetes is constantly growing and a treatment with autologous insulin producing cells would hold great promise for the therapy of diabetes. We herein describe the generation of insulin producing »Neo-islet cells« from human peripheral blood monocytes, which are able to secret insulin in a glucose dependent fashion and normalize blood glucose levels after transplantation into diabetic mice for a period of ten days. In comparison with pancreatic islet cells »Neo-islet cells« exhibit a gene expression pattern which is comparable to an endocrine precursor cell as known from embryonic pancreas developmental studies.
Background: Adoptive cell immunotherapies for opportunistic virus in immunocompromised patients u... more Background: Adoptive cell immunotherapies for opportunistic virus in immunocompromised patients using haploidentical memory T cells have shown to be safe and effective. Since severe cases of COVID-19 present a dysregulated immune system with T cell lymphopenia and a hyper-inflammatory state we have proposed that a similar strategy could be proven to be efficient for COVID-19 patients. This is a study protocol of an open-label, multicenter, double-arm, randomized, dose-finding phase I/II clinical trial to evaluate the feasibility, safety, tolerability, and efficacy of the administration of a single dose of allogenic SARS-CoV-2 specific memory CD45RA - T cells and Natural Killer (NK) cells in COVID-19 patients with lymphopenia and pneumonia. The aim of the study is to find efficient treatments for patients with moderate/severe COVID-19. Identification of Specific memory T cells and NK cells: i)Memory T Cells: we first determined the existence of SARS-CoV-2 specific T cells within the CD45RA - T memory cells of the blood of convalescent donors. Memory T cells can respond quickly to the infection and provide long-term immune protection to reduce the severity of the COVID-19 symptoms without inducing classically T cell alloreactivity. Also, CD45RA - memory T cells confer protection for other pathogens the donors encountered in their life. ii)NK cells: we determined the phenotype of NK cells after COVID-19 and the main characteristic of SARS-CoV-2 specific NK population in the blood of convalescent donors, as it has been shown for cytomegalovirus infections. Also, NK cells confer protection for other pathogens the donors encountered in their life. Pilot Phase I- Safety, feasibility, and dose escalation: Between September and November 2020 a phase 1, dose-escalation, single-center clinical trial was conducted to evaluate the safety and feasibility of the infusion of CD45RA - memory T cells containing SARS-CoV-2 specific T cells as adoptive cell therapy against moderate/severe cases of COVID-19. Nine participants with pneumonia and/or lymphopenia and with at least one human leukocyte antigen (HLA) match with the donor were infused. The first three subjects received the lowest dose (1x10 5 cells/kg), the next three received the intermediate dose (5x10 5 cells/kg) and the last three received the highest dose (1x10 6 cells/kg) of CD45RA - memory T cells. Clinicaltrials.gov registration: NCT04578210. Findings: All participants' clinical status measured by National Early Warning Score (NEWS) and 7-category point ordinal scales showed improvement six days after infusion. No serious adverse events were reported. Inflammatory parameters were stabilized post-infusion and the participants showed lymphocyte recovery two weeks after the procedure. Donor microchimerism was observed at least for three weeks after infusion in all patients. Interpretation: This study provides preliminary evidence supporting the idea that treatment of COVID-19 patients with moderate/severe symptoms using convalescent SARS-CoV-2 specific CD45RA - memory T cells is feasible and safe. We did not find dose-liming toxicity. The Recommended Phase 2 dose was 1x10 6 CD45RA - T cells. Phase II- Efficacy: Between January 2021 and July 2021 patients have been enrolled based on the matched with the HLA genotype of the convalescent donors and following the protocol inclusion/exclusion criteria. The primary outcome is the incidence of patient recovery at day 14, defined as normalization of fever and oxygen saturation or lymphopenia recovery. Secondary outcomes are the time to normal level of lymphocytes, the proportion of patients showing clinical improvement at day 7, time to first negative SARS-CoV-2 PCR, the incidence of treatment-related adverse events, duration of hospitalization, time to discharge, time to improvement by one category a 7-point ordinal scale or NEWS score, the proportion of patients requiring intensive care unit, and all-cause mortality. In addition, lymphocyte recovery by multiparametric flow cytometry and donor chimerism by real-time PCR in the experimental arm was monitored weekly during the first month. This study provides preliminary evidence supporting the idea that treatment of COVID-19 patients with moderate/severe symptoms using convalescent CD45RA - memory T cells is safe and feasible. The phase II clinical trial is ongoing to demonstrate efficacy. Figure 1 Figure 1. Disclosures Soria: Celgene: Other: Fees; Gilead: Other: Fees; AbbVie: Other: Fees.
Aims/hypothesis Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical that has been ass... more Aims/hypothesis Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical that has been associated with type 2 diabetes development. Low doses of BPA modify pancreatic beta cell function and induce insulin resistance; some of these effects are mediated via activation of oestrogen receptors α (ERα) and β (ERβ). Here we investigated whether low doses of BPA regulate the expression and function of ion channel subunits involved in beta cell function. Methods Microarray gene profiling of isolated islets from vehicle-and BPA-treated (100 μg/kg per day for 4 days) mice was performed using Affymetrix GeneChip Mouse Genome 430.2 Array. Expression level analysis was performed using the normalisation method based on the processing algorithm 'robust multi-array average'. Whole islets or dispersed islets from C57BL/ 6J or oestrogen receptor β (ERβ) knockout (Erβ −/−) mice were treated with vehicle or BPA (1 nmol/l) for 48 h. Whole-cell patchclamp recordings were used to measure Na + and K + currents. mRNA expression was evaluated by quantitative real-time PCR. Results Microarray analysis showed that BPA modulated the expression of 1440 probe sets (1192 upregulated and 248 downregulated genes). Of these, more than 50 genes, including Scn9a, Kcnb2, Kcnma1 and Kcnip1, encoded important Na + and K + channel subunits. These findings were confirmed by quantitative RT-PCR in islets from C57BL/6J BPA-treated mice or whole islets treated ex vivo. Electrophysiological measurements showed a decrease in both Na + and K + currents in BPA-treated islets. The pharmacological profile indicated that BPA reduced currents mediated by voltage-activated K + channels (K v 2.1/2.2 channels) and large-conductance Ca 2+-activated K + channels (K Ca 1.1 channels), which agrees with BPA's effects on gene expression. Beta cells from ERβ −/− mice did not present BPA-induced changes, suggesting that ERβ mediates BPA's effects in pancreatic islets. Finally, BPA increased burst duration, reduced the amplitude of the action potential and enlarged the action potential halfwidth, leading to alteration in beta cell electrical activity.
Frontiers in Cell and Developmental Biology, Feb 15, 2023
Type 1 diabetes mellitus (T1DM) is an autoimmune disorder specifically targeting pancreatic islet... more Type 1 diabetes mellitus (T1DM) is an autoimmune disorder specifically targeting pancreatic islet beta cells. Despite many efforts focused on identifying new therapies able to counteract this autoimmune attack and/or stimulate beta cells regeneration, TD1M remains without effective clinical treatments providing no clear advantages over the conventional treatment with insulin. We previously postulated that both the inflammatory and immune responses and beta cell survival/regeneration must be simultaneously targeted to blunt the progression of disease. Umbilical cord-derived mesenchymal stromal cells (UC-MSC) exhibit anti-inflammatory, trophic, immunomodulatory and regenerative properties and have shown some beneficial yet controversial effects in clinical trials for T1DM. In order to clarify conflicting results, we herein dissected the cellular and molecular events derived from UC-MSC intraperitoneal administration (i.p.
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Papers by Bernat Soria